Identifying the molecular drivers of ALS-implicated missense mutations.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressively fatal, neurodegenerative disease associated with both motor and non-motor symptoms, including frontotemporal dementia. Approximately 10% of cases are genetically inherited (familial ALS), while the majority are sporadic. Mutations across a wide range of genes have been associated; however, the underlying molecular effects of these mutations and their relation to phenotypes remain poorly explored. METHODS: We initially curated an extensive list (n=1343) of missense mutations identified in the clinical literature, which spanned across 111 unique genes. Of these, mutations in genes SOD1, FUS and TDP43 were analysed using in silico biophysical tools, which characterised changes in protein stability, interactions, localisation and function. The effects of pathogenic and non-pathogenic mutations within these genes were statistically compared to highlight underlying molecular drivers. RESULTS: Compared with previous ALS-dedicated databases, we have curated the most extensive missense mutation database to date and observed a twofold increase in unique implicated genes, and almost a threefold increase in the number of mutations. Our gene-specific analysis identified distinct molecular drivers across the different proteins, where SOD1 mutations primarily reduced protein stability and dimer formation, and those in FUS and TDP-43 were present within disordered regions, suggesting different mechanisms of aggregate formation. CONCLUSION: Using our three genes as case studies, we identified distinct insights which can drive further research to better understand ALS. The information curated in our database can serve as a resource for similar gene-specific analyses, further improving the current understanding of disease, crucial for the development of treatment strategies.

Searching for plant-derived antivirals against dengue virus and Zika virus.

BACKGROUND: The worldwide epidemics of diseases as dengue and Zika have triggered an intense effort to repurpose drugs and search for novel antivirals to treat patients as no approved drugs for these diseases are currently available. Our aim was to screen plant-derived extracts to identify and isolate compounds with antiviral properties against dengue virus (DENV) and Zika virus (ZIKV). METHODS: Seven thousand plant extracts were screened in vitro for their antiviral properties against DENV-2 and ZIKV by their viral cytopathic effect reduction followed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, previously validated for this purpose. Selected extracts were submitted to bioactivity-guided fractionation using high- and ultrahigh-pressure liquid chromatography. In parallel, high-resolution mass spectrometric data (MSn) were collected from each fraction, allowing compounds into the active fractions to be tracked in subsequent fractionation procedures. The virucidal activity of extracts and compounds was assessed by using the plaque reduction assay. EC50 and CC50 were determined by dose response experiments, and the ratio (EC50/CC50) was used as a selectivity index (SI) to measure the antiviral vs. cytotoxic activity. Purified compounds were used in nuclear magnetic resonance spectroscopy to identify their chemical structures. Two compounds were associated in different proportions and submitted to bioassays against both viruses to investigate possible synergy. In silico prediction of the pharmacokinetic and toxicity (ADMET) properties of the antiviral compounds were calculated using the pkCSM platform. RESULTS: We detected antiviral activity against DENV-2 and ZIKV in 21 extracts obtained from 15 plant species. Hippeastrum (Amaryllidaceae) was the most represented genus, affording seven active extracts. Bioactivity-guided fractionation of several extracts led to the purification of lycorine, pretazettine, narciclasine, and narciclasine-4-O-ß-D-xylopyranoside (NXP). Another 16 compounds were identified in active fractions. Association of lycorine and pretazettine did not improve their antiviral activity against DENV-2 and neither to ZIKV. ADMET prediction suggested that these four compounds may have a good metabolism and no mutagenic toxicity. Predicted oral absorption, distribution, and excretion parameters of lycorine and pretazettine indicate them as candidates to be tested in animal models. CONCLUSIONS: Our results showed that plant extracts, especially those from the Hippeastrum genus, can be a valuable source of antiviral compounds against ZIKV and DENV-2. The majority of compounds identified have never been previously described for their activity against ZIKV and other viruses.

Variant type is associated with disease characteristics in SDHB, SDHC and SDHD-linked phaeochromocytoma-paraganglioma.

BACKGROUND: Pathogenic germline variants in subunits of succinate dehydrogenase (SDHB, SDHC and SDHD) are broadly associated with disease subtypes of phaeochromocytoma-paraganglioma (PPGL) syndrome. Our objective was to investigate the role of variant type (ie, missense vs truncating) in determining tumour phenotype. METHODS: Three independent datasets comprising 950 PPGL and head and neck paraganglioma (HNPGL) patients were analysed for associations of variant type with tumour type and age-related tumour risk. All patients were carriers of pathogenic germline variants in the SDHB, SDHC or SDHD genes. RESULTS: Truncating SDH variants were significantly over-represented in clinical cases compared with missense variants, and carriers of SDHD truncating variants had a significantly higher risk for PPGL (p<0.001), an earlier age of diagnosis (p<0.0001) and a greater risk for PPGL/HNPGL comorbidity compared with carriers of missense variants. Carriers of SDHB truncating variants displayed a trend towards increased risk of PPGL, and all three SDH genes showed a trend towards over-representation of missense variants in HNPGL cases. Overall, variant types conferred PPGL risk in the (highest-to-lowest) sequence SDHB truncating, SDHB missense, SDHD truncating and SDHD missense, with the opposite pattern apparent for HNPGL (p<0.001). CONCLUSIONS: SDHD truncating variants represent a distinct group, with a clinical phenotype reminiscent of but not identical to SDHB. We propose that surveillance and counselling of carriers of SDHD should be tailored by variant type. The clinical impact of truncating SDHx variants is distinct from missense variants and suggests that residual SDH protein subunit function determines risk and site of disease.

Identification and in silico characterization of structural and functional impacts of genetic variants in milk protein genes in the Zebu breeds Guzerat and Gyr.

Whole genome sequencing of bovine breeds has allowed identification of genetic variants in milk protein genes. However, functional repercussion of such variants at a molecular level has seldom been investigated. Here, the results of a multistep Bioinformatic analysis for functional characterization of recently identified genetic variants in Brazilian Gyr and Guzerat breeds is described, including predicted effects on the following: (i) evolutionary conserved nucleotide positions/regions; (ii) protein function, stability, and interactions; (iii) splicing, branching, and miRNA binding sites; (iv) promoters and transcription factor binding sites; and (v) collocation with QTL. Seventy-one genetic variants were identified in the caseins (CSN1S1, CSN2, CSN1S2, and CSN3), LALBA, LGB, and LTF genes. Eleven potentially regulatory variants and two missense mutations were identified. LALBA Ile60Val was predicted to affect protein stability and flexibility, by reducing the number the disulfide bonds established. LTF Thr546Asn is predicted to generate steric clashes, which could mildly affect iron coordination. In addition, LALBA Ile60Val and LTF Thr546Asn affect exonic splicing enhancers and silencers. Consequently, both mutations have the potential of affecting immune response at individual level, not only in the mammary gland. Although laborious, this multistep procedure for classifying variants allowed the identification of potentially functional variants for milk protein genes.

Tumour risks and genotype-phenotype correlations associated with germline variants in succinate dehydrogenase subunit genes SDHB, SDHC and SDHD.

BACKGROUND: Germline pathogenic variants in SDHB/SDHC/SDHD are the most frequent causes of inherited phaeochromocytomas/paragangliomas. Insufficient information regarding penetrance and phenotypic variability hinders optimum management of mutation carriers. We estimate penetrance for symptomatic tumours and elucidate genotype-phenotype correlations in a large cohort of SDHB/SDHC/SDHD mutation carriers. METHODS: A retrospective survey of 1832 individuals referred for genetic testing due to a personal or family history of phaeochromocytoma/paraganglioma. 876 patients (401 previously reported) had a germline mutation in SDHB/SDHC/SDHD (n=673/43/160). Tumour risks were correlated with in silico structural prediction analyses. RESULTS: Tumour risks analysis provided novel penetrance estimates and genotype-phenotype correlations. In addition to tumour type susceptibility differences for individual genes, we confirmed that the SDHD:p.Pro81Leu mutation has a distinct phenotype and identified increased age-related tumour risks with highly destabilising SDHB missense mutations. By Kaplan-Meier analysis, the penetrance (cumulative risk of clinically apparent tumours) in SDHB and (paternally inherited) SDHD mutation-positive non-probands (n=371/67 with detailed clinical information) by age 60 years was 21.8% (95% CI 15.2% to 27.9%) and 43.2% (95% CI 25.4% to 56.7%), respectively. Risk of malignant disease at age 60 years in non-proband SDHB mutation carriers was 4.2%(95% CI 1.1% to 7.2%). With retrospective cohort analysis to adjust for ascertainment, cumulative tumour risks for SDHB mutation carriers at ages 60 years and 80 years were 23.9% (95% CI 20.9% to 27.4%) and 30.6% (95% CI 26.8% to 34.7%). CONCLUSIONS: Overall risks of clinically apparent tumours for SDHB mutation carriers are substantially lower than initially estimated and will improve counselling of affected families. Specific genotype-tumour risk associations provides a basis for novel investigative strategies into succinate dehydrogenase-related mechanisms of tumourigenesis and the development of personalised management for SDHB/SDHC/SDHD mutation carriers.

Correction: Variation in Human Cytochrome P-450 Drug-Metabolism Genes: A Gateway to the Understanding of Plasmodium vivax Relapses.

[This corrects the article DOI: 10.1371/journal.pone.0160172.].

Variation in Human Cytochrome P-450 Drug-Metabolism Genes: A Gateway to the Understanding of Plasmodium vivax Relapses.

Although Plasmodium vivax relapses are classically associated with hypnozoite activation, it has been proposed that a proportion of these cases are due to primaquine (PQ) treatment failure caused by polymorphisms in cytochrome P-450 2D6 (CYP2D6). Here, we present evidence that CYP2D6 polymorphisms are implicated in PQ failure, which was reinforced by findings in genetically similar parasites, and may explain a number of vivax relapses. Using a computational approach, these polymorphisms were predicted to affect the activity of CYP2D6 through changes in the structural stability that could lead to disruption of the PQ-enzyme interactions. Furthermore, because PQ is co-administered with chloroquine (CQ), we investigated whether CQ-impaired metabolism by cytochrome P-450 2C8 (CYP2C8) could also contribute to vivax recurrences. Our results show that CYP2C8-mutated patients frequently relapsed early (<42 days) and had a higher proportion of genetically similar parasites, suggesting the possibility of recrudescence due to CQ therapeutic failure. These results highlight the importance of pharmacogenetic studies as a tool to monitor the efficacy of antimalarial therapy.